This invention relates to processes for preparing water soluble poly(ethylene glycol) conjugates of macrolide immunosuppressants sirolimus (rapamycin), everolimus, temsirolimus (CCI-779), tacrolimus (FK506) and ascomycin (FK520).
Rapamycin (1), tacrolimus (FK506, 2) and ascomycin (FK520, 3) are structurally similar macrocyclic polyketides and all are potent immunosuppressants that interact with the same intracellular receptors, but have different modes of action, suppressing T-cell activation at different stages (Rosen et al., “Natural products as probes of cellular function: studies of immunophilins” Angew. Chem. Int. Ed. Engl. 1992, 31, 384-400). These macrolides have antimicrobial activity and are also effective in animal models of autoimmune diseases including experimental allergic encephalomyelitis, arthritis, animal models of diabetes, the MRL/lpr mouse model of SLE, hyperproliferative ski diseases, and uveoretinitis.

Rapamycin and tacrolimus have been approved for preventing transplantation rejection. However, both compounds share similar problems in formulating the compositions due to their very limited aqueous solubility. For example, tacrolimus has a solubility of 12 μg/mL in water. Such a low solubility requires a rather complicated formulation. For instance, 200 mg/mL of hydrogenated polyoxy 60 castor oil (HCO-60) and 80% (v/v) absolute alcohol are required as the solubilizing aid for dissolving 5 mg of tacrolimus for intravenous injections. Rapamycin has a solubility of about 2.6 μg/mL in water and low oral bioavailability (<15%) (Yatscoff et al., “Rapamycin: distribution, pharmacokinetics, and therapeutic range investigations” Ther. Drug Monit. 1995, 17, 666-671). These characteristics have limited rapamycin's clinical applications other than low-dosage treatment such as immunosuppression, despite it is also a potent inhibitor of tumor growth with a typical IC50<50 nm against various solid tumors.
Polyethylene glycol (PEG) and methoxy polyethylene glycol (mPEG) are linear or branched, neutral polymers available in a variety of molecular weights with low polydispersities (Mw/Mn<1.05). These water/organic solvent soluble, non-toxic polymers have been found useful in biological and pharmaceutical applications. One such application is the binding of these polymers with the non or sparingly water-soluble small molecule therapeutics to make water soluble PEG-drug conjugates, termed PEGylation. Pegylation of organic molecules has been reported to enhance aqueous solubility of the organic molecule and to confer other beneficial properties such as improved plasma half-life, improved biological distribution, and reduced toxicity (Greenwald et al., “Effective drug delivery by PEGylated drug conjugates”, Advanced Drug Delivery Rev. 2003, 55, 217-250; Pasut et al., “Protein, peptide and non-peptide drug PEGylation for therapeutic application”, Expert Opin. Ther. Patents 2004, 14, 859-894).
The lipase-catalyzed acetylation of rapamycin has been discussed in US Patent Application Publication No. US-2005/0234234, which is hereby incorporated by reference. This enzymatic process gives rapamycin 42-ester derivatives regiospecifically from rapamycin with excellent yield under mild condition.
The preparation of PEG conjugates of rapamycin or its derivatives has been described in U.S. Pat. Nos. 5,955,457; 5,780,462; 6,432,973 and 6,331,547. The preparation of hydroxyester of rapamycin CCI-779, from which the pegylated CCI-779 was made from, was described in the U.S. Pat. No. 5,362,718, which is hereby incorporated by reference. These patents describe conjugates formed by chemically linking rapamycin or its derivatives to methoxy polyethylene glycol compounds such as a thiol derivative (mPEGSH) through an ester linkage. Solvent extraction and chromatography purification were thereby required to recover the desired PEG conjugate. By doing so, rapamycin 42-iodoacetate was prepared in a 55% yield after high performance liquid chromatography (HPLC) purification.
The preparation of the water soluble PEG-tacrolimus (FK-506) conjugate is discussed in International Patent Publication No. WO 99/03860 using a similar procedure. One major drawback of these procedures is the low selectivity of installation of the ester linkage due to the presence of multiple OH-functionalities in rapamycin/tacrolimus skeleton. Additionally, the use of aqueous sodium hydrogen carbonate as a base during pegylation generates several by-products, requiring multiple purification steps with low or moderate recovery yield.
The synthesis of everolimus is described in the U.S. Pat. No. 6,440,990 and the synthesis of PEG-everolimus (II) was described in the U.S. Pat. No. 6,331,547, which patents are hereby incorporated by reference.
What are needed in the art are alternate processes for preparing water soluble poly(ethylene glycol) conjugates of macrolide immunosuppressants.